Process of obtaining type II dehydroquinase enzyme inhibitors and precursors thereof

ABSTRACT

The present invention relates to a process of obtaining type II dehydroquinase enzyme inhibitors and the precursors thereof from (-)-quinic acid. The described compounds have a carboxycyclohexene structure. The process of preparing the compounds and their application as compositions with pharmacological properties and herbicides of interest are described.

The present invention relates to the process of obtaining type IIdehydroquinase enzyme inhibitors having a carboxycyclohexene structure,and to obtaining the intermediate precursors of said inhibitors. Thecompounds are prepared from (−)-quinic acid.

The inhibitors have the following formula:

where R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ can be hydrogen, alkyloxy or alkylwith C1-C10 chains, or any aromatic compound, or a benzyloxy group inwhich the aromatic ring can be substituted by one or severalsubstituents chosen from halogen, nitro, guanidinium, azido, cyano,phosphate, amino, carboxy, amide, thiol, thioester, thioether, alcohol,alkoxy or alkyl groups with C1-C10 chains.

In the field of agriculture, the growth of undesirable weeds togetherwith the crops creates significant problems, such as the decrease inproduction and quality, farm work and harvesting difficulties, and theneed for manual labor or pesticides for their elimination. For thisreason, unwanted weeds limit agricultural production and considerablyaffect their price. It is estimated that the decrease in productionbrought about by the growth of weeds is 15-20% the total crop value,mainly due to the competition of the weeds with the useful plants forthe consumption of nutritive elements, water and light. As a result theuse of herbicides has been imposed as one of the necessary operationsfor achieving stable, high-yield crops. The current treatment withherbicides is so extensive that it has given rise to a very importantbranch of the chemical industry.

It is necessary to discover new herbicides with maximum selectivity,such that they preferably attack weeds, minimally affecting the crops.Selectivity is often achieved by means of chemicals interfering inbiogenetic pathways present in the weeds. Hence there are herbicidesinterfering in the biosynthesis of proteins, aromatic amino acids,lipids or carotenoids.

Chorismic acid is a key intermediate in the biosynthesis of the aromaticamino acids tyrosine, phenylalanine and tryptophan:

Chorismic acid is also a key intermediate in the biosynthesis of otherbiologically important products such as: p-aminobenzoate, folic acid,p-hydroxybenzoate and certain vitamins:

Chorismic acid is in turn biosynthesized by means of a series ofchemical reactions known with the name of the shikimic acid pathway (fora review on this topic please see (a) Abell, C.Enzymology and MolecularBiology of the Shikimate Pathway: Comprehensive Natural ProductsChemistry, Sankawa, U.; Pergamon, Elsevier Science Ltd. Oxford, 1999,Vol 1, page 573; (b) Haslam, E. Shikimic Acid: Metabolism andMetabolites, John Wiley, Chichester, 1993). This biosynthetic pathway ispresent in the secondary metabolism of plants, fungi and bacteria, butnot in animals (Hawkins, A. R. CRC Crit. Rev. Biochem. 1990, 25, 307),so it is considered a very important source for the development of newherbicides, fungicides or antibiotics capable of selectively blockingcertain enzymatic transformations of this biosynthetic pathway (pleasesee: (1) Jaworski, E. G. Food Chem. 1972, 20, 1195. (b) Baillie, A. C.;Corbett, J. R.; Dowsett, J. R.; McCloskey, P. Pestic. Sci. 1972, 3, 113.(c) Kishore, G. M.; Shah, D. M. Annun. Rev. Biochem. 1988, 57, 627). Itmust be taken into account that an herbicide acting on a metabolicpathway present in plants but not in animals is expected to presentminimum toxicity in humans. The best herbicide currently producedworld-wide, Glyphosphate, acts precisely by inhibiting the sixth enzymeof the shikimic acid pathway (EPSP synthase) with a magnificentinhibition constant of about 1 μM (please see: (a) Steinrucken, H. C.;Amerhein, N. Eur. J. Biochem. 1984, 143,351. (b) Steinrucken, H. C.;Amerhein, N. Biochem. Biophys. Res. Commun. 1980, 94, 1207).Glyphosphate forms a complex with the enzyme and 3-phosphate shikimatewhich inhibits enzymatic activity and is considered to be responsiblefor the herbicidal activity thereof. Glyphosphate is the activecomponent of the Roundup and Tumbleweed herbicides widely used asselective, post-emergent low toxicity herbicides.

It must be further stressed that recently Roberts et al. (Nature 1998,393, 801) demonstrated the surprising presence of these enzymes of theshikimic acid pathway in certain animal parasites of the Phylumapicomplexa, such as the Toxoplasma gondii, Plasmodium falciparum(malaria) and Cryptosporidium parvum. Therefore herbicides inhibitingthe shikimic acid pathway may be effective against these organisms. Infact, it has been proven that glyphosphate is effective against malaria(see: (a) McFadden, G. I.; Keith, M. E.; Monholland, J. M.; Lang-Unasch,N. Nature 1996, 381, 482; (b) Fichera, M. E.; Roos, D. S. Nature 1997,390, 407). This disease together with AIDS and tuberculosis, form themost deadly trio of infectious diseases for mankind as of todayaccording to the World Health Organization. Therefore it is possiblethat compounds with herbicidal properties may additionally haveantimalarial activity.

It is therefore possible to obtain compounds with a broad activityspectrum. The considerable interest in them is based on the fact thatthey can be used in the treatment of diseases caused by severalpathogenic agents simultaneously infecting a living being.

A selective and effective herbicide inhibiting some of the enzymespresent in the shikimic acid pathway can be obtained. The interest ofthe inventors is focused on the third enzyme of this biosyntheticpathway, dehydroquinase (3-dehydroquinate dehydratase, EC 4.2.1.10),which catalyzes 3-dehydroquinic acid dehydration to 3-dehydroshikimicacid. Two types of dehydroquinases, referred to as type I and type II,are known due to their different biophysical properties and thedifferent amino acid sequence they have (see Hawkins, A. R. Curr. Genet.1987, 11, 491). Both types catalyze the same transformation but by meansof different mechanisms and with opposite stereochemistry (seeKleanthous, C.; Davis, K.; Kelly, S. M.; Cooper, A.; Harding, S. E.;Price, N. C.; Hawkins, A. R.; Coggins, J. R. Biochem. J. 1992, 282,687). Type II dehydroquinases, which come from different sources(Mycobacterium tuberculosis, Streptomyces coelicolor and Aspergillusnidulans) are dodecameric (12-16 KDa) and thermally stable, whereas thetype I enzymes are dimeric (27 KDa) and temperature-sensitive.

The most studied enzyme is dehydroquinase type I, from Escherichia coli(see Chauduri, C.; Ducan, K.; Graham, L. D.; Coggins, J. R. Biochem. J.1990, 275, 1). Coggins et al. (J. Am. Chem. Soc. 1991, 113, 9416; J.Biol. Chem. 1995, 270, 25827) proved that its mechanism of action occursthrough the formation of a Schiff base between the ketone group and alysine residue in the active center (Lys170). This entails the loss ofthe pro-R hydrogen at C-2, globally corresponding to an elimination ofwater in syn (see: (a) Hanson, K. R.; Rose, I. A. Proc. Natl. Acad. Sci.USA 1963, 50, 981; (b) Smith, B. W.; Turner, M. J.; Haslam, E. J. Chem.Soc., Chem. Commun. 1970, 842; (c) Haslam, E.; Turner, M. J.; Sargent,D.; Thompson, R. S. J. Chem. Soc. (C) 1971, 1489).

In contrast, the type II enzyme (see: (a) Gourley, D. G.; Coggins, J.R.; Isaacs, N. W.; Moore, J. D.; Charles, I. G.; Hawkins, A. R. J. Mol.Biol. 1994, 241, 488; (b) Krell, T.; Pilt, A. R.; Coggins, J. R. FEBSLett. 1995, 360, 93) catalyzes the elimination of water in anti with theloss of the hydrogen plus acid, the pro-S (see: (a) Shneier, A.; Harris,J.; Kleanthous, C.; Coggins, J. R.; Hawkins, A. R.; Abell, C. Bioorg.Med. Chem. Lett. 1993, 3, 1399; (b) Harris, J.; Kleanthous, C.; CogginsJ. R.; Hawkins, A. R.; Abell, C. J. Chem. Soc., Chem. Commun. 1993, 13,1080). Abell et al. proposed that the reaction occurs through an E1CBmechanism through an intermediate enolate (Biochem. J. 1996, 319, 333).

Recently, Lapthorn et al. (Structure 2002, 10, 493) were able to resolvethe crystalline structure of the type II dehydroquinase fromStreptomyces coelicolor. This X-ray structure has allowed clearlydefining both the position and the structure of the active center. Andmore importantly it has clarified the role that the amino acids of theactive center play as well as confirmed the enolic mechanism previouslyproposed by Abell.

Lapthorn et al. propose that Tyr28 acts as a base in the first step ofabstraction of the axial proton at alpha to the ketone. It must bepointed out that this Tyr residue is deprotonated due to the basicenvironment in which it is located and which Arg113 is responsible for.After deprotonation, they propose that the substrate forms an enolateintermediate. And although there is no residue that is close enough tostabilize the negative charge, a water molecule is correctly located inits place at 2.8 Å from this group coordinated with the amide group ofAsn16, the carbonyl of Pro15 and the nitrogen of Ala82. The eliminationof water finally occurs, which step is catalyzed by His106 acting as aproton donor, and the carbonyl of Asn79 also acting as a protonaccepter, favoring the final elimination of water.

A class of compounds is described in the present invention that ischaracterized by having a six-membered ring with a double bond betweenpositions 5 and 6 and a carboxylic group at position 1. Objectives ofthis invention are compounds with the hereinbefore mentioned basicstructure in which the R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ groups can behydrogen, alkyloxy, alkyl with C1-C10 chains, or any aromatic compound,or a benzyloxy group in which the aromatic ring may be substituted byone or several identical or different radicals, chosen from halogen,polyhalogenated alkyl, nitro, azido, amino, phosphate, carboxy, amide,thiol, thioester, guanidinium, thioether, alcohol, alkoxy or alkylgroups with C1-C10 chains.

In all of them, the different substituents are radicals of the followingtype: linear or branched alkyl with 1-10 carbon atoms, alkenyl with 2 to10 carbon atoms, alkynyl with 3 to 10 carbon atoms, cycloalkyl with 3 to6 carbon atoms, cycloalkenyl with 4 to 6 carbon atoms, or bicycloalkylwith 7 to 10 carbon atoms; these radicals possibly being substituted byone or several identical or different substituents chosen from halogenatoms and the hydroxy, amino, thiol, azido, nitro, phosphate and alkoxyradicals containing 1 to 4 carbon atoms, piperidinyl, morpholinyl,indole, furan, piperazinyl-1 (possibly substituted at −4 by an alkylradical with 1 to 4 carbon atoms or by a phenylalkyl radical, the alkylpart of which contains from 1 to 4 carbon atoms), cycloalkyl with 3 to 6carbon atoms, cycloalkenyl with 4 to 6 carbon atoms, phenyl, cyano,nitro, carboxy, alkoxycarbonyl, halogen, amino or amide, the alkyl partof which contains 1 to 4 carbon atoms, or a phenyl radical, possiblysubstituted by one or several identical or different radicals, chosenfrom the alkyl radicals with 1 to 4 carbon atoms, halogenated or not, oran alkoxy with 1 to 4 carbon atoms, or halogen, nitro, azido, phosphate,amino, cyano, amide, thiol, thioester, guanidinium, thioether or alcoholgroups, a saturated or unsaturated nitrogenous heterocyclic radicalcontaining 1 to 4 carbon atoms, a saturated or unsaturated nitrogenousheterocyclic radical containing 5 or 6 members, possibly substituted byone or several alkyl radicals with 1 to 4 carbon atoms, understandingthat the cycloalkyl, cycloalkenyl or bicycloalkyl radicals can possiblybe substituted by one or several alky radicals containing 1 to 4 carbonatoms.

Also object of the present invention are the herbicidal andpharmaceutical properties of the compounds hereinbefore mentioned,including their anticancerous and antibiotic properties. The process ofobtaining said compounds is finally described.

The process of obtaining these compounds is based on the chemicalmodification by means of simple transformations of a basic skeleton,either by means of solution chemistry or by means of solid supportchemistry. The key steps of these transformations consist of thealkylation of the alkoxides derived from these basic compounds, suitablyfunctionalized, either in a solution or a solid support with differentelectrophiles; the cleaving of the compounds from the resin for the caseof solid support synthesis, and finally the hydrolysis reaction leadingto obtaining the acid group.

EXAMPLE 1

The cyclohexene acids (VI) were prepared following the mentioned solidphase synthetic strategy and using carbolactone I, BromoWang polystyreneresin and the corresponding benzyl bromide derivative as startingmaterials.

(a) K₂CO₃, MeOH, 60° C., 67%; (b) 1. HNa, DMF, O° C.; 2. BromoWangresin, 15-crown-5, r.t.; (c) TBAF, THF, r.t.; (d) 1. HNa, DMF, 0° C.; 2.R-phenyl-CH₂Br, 15-crown-5, 40-80° C.; (e) 1. 50% TFA/DCM, 2. LiOH, THF,r.t., 3. Amberlite IR-120 (H⁺).

Therefore, treatment of carbolactone I with potassium carbonate inmethanol provides allyl alcohol II (see Gonzalez, C.; Carballido, M.;Castedo, L. J. Org. Chem. 2003, 68, 2248) which, by means of treatingwith sodium hydride and subsequent reaction with BromoWang polystyreneresin leads to ether III. Treatment of the ether III withtetrabutylammonium fluoride provides the tertiary alcohol IV which, byreaction with sodium hydride and subsequent reaction with thecorresponding benzyl bromides gives rise to ethers V. The desired acidsV are finally obtained by means of a three-step process first consistingof breaking the bond with the resin by reaction with trifluoroaceticacid, basic hydrolysis of the lactone and finally treatment withAmberlite IR-120, an ion exchange resin.

Preparation of Resin III.

Sodium hydride (222 mg, 5.54 mmol, 60% commercial suspension in mineraloil) was added to a solution of alcohol II (1.4 g, 5.18 mmol) in dry DMF(20 mL) cooled at 0° C. and under an argon atmosphere. After 30 minutesat said temperature, the resulting suspension was added by means of acannula to a suspension of the BromoWang polystyrene resin (1 g, ˜1.6mmol/g) in dry DMF (17 mL) cooled at 0° C. and under an argonatmosphere. Then 15-crown-5 ether (30 μL, 0.26 mmol) was added and theresulting suspension was gently stirred at 0° C. for 30 minutes and atroom temperature for 24 hours. The resin was filtered and successivelywashed with DMF (3×15 mL), (3:1) DMF/water (3×15 mL), THF (3×15 mL) anddichloromethane (3×15 mL). 1.02 g of resin III were obtained in a paleyellow granule form after vacuum-drying. IR (gel/cm⁻¹) 1797 and 1611;¹³C-NMR (gel, 63 MHz, CDCL₃, δ) 128.5, 118.7, 114.7, 73.7, 71.2, 69.9,64.8, 40.0, 37.7, 25.6 and −3.1.

Preparation of Resin IV.

Tetrabutylammonium fluoride (1.4 mL, 1M commercial solution in THF) wasadded to a suspension of resin III (1 g, ˜1 mmol) in dry THF (16 mL) at0° C. and under inert atmosphere. The resulting suspension was gentlystirred for 2 hours at room temperature. The resin was filtered andwashed successively with THF (3×15 mL), (3:1) 5% HCl/THF (3×15 mL), THF(3×15 mL) and dichloromethane (3×15 mL). 0.9 g of resin IV were obtainedin the form of pale yellow grains after vacuum-drying. IR (gel/cm⁻¹)3414, 1789 and 1609; ¹³C-NMR (gel, 63 MHz, CDCL₃, δ) 136.4, 129.5,121.5, 115.3, 74.6, 71.1, 53.5 and 36.9.

General Alkylation Process.

A suspension of resin IV in dry DMF (1 mL for every 100 mg of resin) at0° C. and under inert atmosphere was treated with 6 equivalents ofsodium hydride (60% commercial suspension in mineral oil). The resultingsuspension was gently stirred for 1 hour at room temperature and then 10equivalents of the corresponding benzyl bromide and 0.3 equivalents of15-crown-5 ether were added. The resulting suspension was heated between40-80° C. for 24 to 48 hours. The resin was filtered and washed with THF(3×), (3:1) 10% HCl/THF (3×) and dry dichloromethane (3×) to give riseto resin V.

General Process of Breaking the Resin Compounds.

The resin was treated at room temperature for 1 hour with a 50%TFA/dichloromethane mixture (1 mL for every 100 mg of resin). The resinwas filtered and washed with dichloromethane (3×). The filtrate wasconcentrated under reduced pressure and vacuum-dried for 15 minutes. Theobtained residue was redissolved in THF and treated with 5 equivalentsof a 0.5 M lithium hydroxide aqueous solution. After 30 minutes, milliQwater was added and the aqueous phase was washed with diethyl ether(3×). The aqueous extract was treated with Amberlite IR-120 (H⁺) to pH6. The resin was filtered and washed with milliQ water. The filtrate waslyophilized so as to yield a colorless oil or foam, as appropriate.

The data of some compounds obtained using this process is given below:

(1R,3R,4R)-1-benzyloxy-3,4-dihydroxycyclohex-5-ene-1-carboxylic acid(VIa).

[α]²⁵ _(D)+15° (c 0.7 in H₂O); ¹H-NMR (250 MHz, D₂O, δ) 7.31 (m, 5H),5.93 (d, 1H, J 10.1), 5.82 (dd, 1H, J 10.1 and 1.8), 4.41 (s, 2H), 4.00(dd, 1H, J 8.3 and 1.8), 3.77 (td, 1H, J 8.3, 11.9 and 3.5), 2.14 (dd,1H, J 13.6 and 3.5) and 1.87 (t, 1H, J 13.6 and 11.9); ¹³C-NMR (100 MHz,D₂O, δ) 178.1, 137.9, 134.2, 128.8, 128.8, 128.4, 127.3, 80.8, 72.6,69.5, 67.7 and 37.9; IR (KBr)/cm⁻¹ 3434, 1714 and 1578; EM-IQ⁺ (m/z) 247(MH⁺-H₂O); HRMS calculated for C₁₄H₁₅O₄ (MH⁺): 247.0970, 247.0965 found.

(1R,3R,4R)-1-(2′-fluorobenzyloxy)-3,4-dihydroxycyclohex-5-ene-1-carboxylicacid (VIb).

[α]²⁵ _(D)−5° (c 0.7 in H₂O); ¹⁹F-NMR (282 MHz, D₂O, δ) −117.0 (dt, 1F,J 10.5 and 6.3); ¹H-NMR (250 MHz, D₂O, δ) 7.41-7.29 (m, 2H), 7.15-7.02(m, 2H), 5.94 (d, 1H, J 10.1), 5.86 (dd, 1H, J 10.1 and 1.7), 4.50 (s,2H), 4.02 (dt, 1H, J 8.2 and 1.7), 3.78 (ddd, 1H, J 12.1, 3.6 and 8.2),2.18 (ddd, 1H, J 13.7, 3.6 and 1.4) and 1.90 (dd, 1H, J 13.7 and 12.1);¹³C-NMR (63 MHz, D₂O, δ) 177.8, 161.2 (J 244), 135.0, 131.8 (J 4), 130.9(J 8), 127.0, 124.6 (J 18), 124.7, 115.7 (J 21), 80.3, 72.7, 69.6, 61.6(J 4) and 37.8 (CH₂); IR (KBr)/cm⁻¹ 3420, 1717 and 1589; EM-IQ⁺ (m/z)265 (MH⁺-H₂O); HRMS calculated for C₁₄H₁₄O₄F (MH⁺): 265.0876, 265.0876found.

(1R,3R,4R)-3,4-dihydroxy-1-(4′-carboxy)benzyloxycyclohex-5-ene-1-carboxylicacid (VIc).

M.P. 161-162° C.; [α]²⁵ _(D)+16° (c 1.3 in CH₃OH); ¹H-NMR (250 MHz,CD₃OD, δ) 7.94 (d, 2H, J 8.2), 7.45 (d, 2H, J 8.2), 6.01 (d, 1H, J10.1), 5.87 (dd, 1H, J 10.1 and 1.9), 4.61 (d, 1H, J 11.8), 4.54 (d, 1H,J 11.8), 3.99-3.82 (m, 2H), 2.26 (dd, 1H, J 13.2 and 3.4), and 1.96 (dd,1H, J 13.2 and 11.5); ¹³C-NMR (63 MHz, CD₃OD, δ) 177.0, 170.0, 145.6,135.7, 130.6, 128.5, 128.2, 80.7, 74.1, 70.9, 67.5 and 30.8; IR(KBr)/cm⁻¹ 3444 and 1697; EM-IQ⁺ (m/z) 291 (MH⁺-H₂O); HRMS calculatedfor C₁₅H₁₅O₆ (MH⁺): 291.0869, 291.0873 found.

EXAMPLE 2

Cyclohexene acid (IX) was prepared following the mentioned syntheticstrategy in solution, using hydroxycarbolactone II and 4-nitrobenzylbromide as starting materials. Thus, treatment of the sodium alkoxidederivative of hydroxylactone II with 4-nitrobenzyl bromide providesether VII which, by reaction with tetrabutylammonium fluoride, gives thetertiary alcohol VIII. Finally, the basic hydrolysis of lactone VIII andsubsequent treatment with the ion exchange resin Amberlite IR-120provides the desired compound IX.

-   -   (a) 1. HNa, DMF, 0° C.; 2. 4-nitrobenzyl bromide, Bu₄NI,        15-crown-5, 80° C.; (b) TBAF, THF, 0° C.; (c) 1. LiOH, THF,        r.t., 2. Amberlite IR-120 (H⁺).        Preparation of        (1R,3R,4R)-1-(tert-butyldimethylsilyloxy)-4-(4′-nitrobenzyloxy)cyclohex-5-ene-1,3-carbolactone        (VII).

Sodium hydride (29 mg, 0.73 mmol, 60% commercial suspension in mineraloil) was added to a solution of alcohol II (164 mg, 0.61 mmol) in dryDMF (6 mL) at 0° C. and under an argon atmosphere. After 30 minutes atthis temperature, 4-nitrobenzyl bromide (171 mg, 0.79 mmol),tetrabutylammonium iodide (23 mg, 0.06 mmol) and 15-crown-5 ether (10μL, 0.08 mmol) were added. The resulting blue solution was heated at 80°C. for 48 hours. The obtained suspension was diluted with diethyl ether(5 mL) and with water (15 mL). The organic phase was separated and theaqueous phase extracted with diethyl ether (3×20 mL). The pooled organicphase was dried (Na₂SO₄ anh.), filtered and concentrated under reducedpressure. The obtained residue was purified by means of flashchromatography in silica gel (70% dichloromethane/hexanes) to give 60 mg(24%) of ether VII in a pale yellow solid form. [α]²⁵ _(D)−172° (c 2.0in CHCl₃); ¹H-NMR (250 MHz, CDCl₃, δ) 8.21 (d, 2H, J 8.8), 7.50 (d, 2H,J 8.8), 6.15 (ddd, 1H, J 9.8, 1.7 and 1.0), 5.78 (ddd, 1H, J 9.8, 3.2and 1.1), 4.77 (s, 2H), 4.73 (m, 1H), 3.98 (t, 1H, J 3.2), 2.42 (ddd,1H, J 10.6, 5.2 and 1.8), 2.37 (d, 1H, J 10.6), 0.90 (s, 9H), 0.17 (s,3H) and 0.14 (s, 3H); ¹³C-NMR (63 MHz, CDCl₃, δ) 175.3, 147.5, 144.9,138.9, 127.7, 124.2, 123.7, 75.0, 73.4, 72.4, 70.9, 37.7, 25.5, 17.9 and−3.1; IR (NaCl)/cm⁻¹ 1793; EM-IQ⁺ (m/z) 406 (MH⁺); HRMS calculated forC₂₀H₂₈O₆NSi (MH⁺): 406.1686, 406.1676 found.

Preparation of(1R,3R,4R)-1-hydroxy-4-(4′-nitrobenzyloxy)cyclohex-5-ene-1,3-carbolactone(VIII).

Tetrabutylammonium fluoride (0.25 mL, 0.25 mmol, 1.0 M commercialsolution in THF) was added to a solution of silyl ether VII (95 mg, 0.23mmol) in 2 mL of dry THF under an argon atmosphere and at 0° C. Afterstirring for 30 minutes at said temperature, it was acidulated with 10%HCl and the organic phase was extracted with dichloromethane (3×15 mL).The pooled organic phase was dried (Na₂SO₄ anh.), filtered andconcentrated under reduced pressure. The obtained residue was purifiedby means of flash chromatography in silica gel (diethyl ether) to give29 mg of alcohol VIII in a pale yellow oil form (43%). [α]²⁵ _(D)−265.5°(c 1.2 in CHCl₃); ¹H-NMR (250 MHz, CDCl₃, δ) 8.22 (d, 2H, J 8.7), 7.51(d, 2H, J 8.7), 6.17 (d, 1H, J 9.8), 5.82 (ddd, 1H, J 9.8, 3.3 and 1.0),4.81 (m, 1H), 4.78 (s, 2H), 4.02 (t, 1H, J 3.3), 3.35 (broad s, 1H) and2.44 (m, 2H); ¹³C-NMR (63 MHz, CDCl₃, δ) 177.2, 147.6, 144.8, 137.4,127.7, 125.1, 123.8, 74.5, 73.4, 72.2, 71.0 and 36.9; IR (NaCl)/cm⁻¹3405 and 1784; EM-IQ⁺ (m/z) 292 (MH⁺); HRMS calculated for C₁₄H₁₄O₆N(MH⁺): 292.0821, 292.0826 found.

Preparation of(1R,3R,4R)-1,3-dihydroxy-4-(4′-nitrobenzyloxy)cyclohex-5-ene-1-carboxylicacid (IX).

A solution of carbolactone VIII (28 mg, 0.10 mmol) in 1 mL of THF and0.5 mL of a 0.5 M lithium hydroxide aqueous solution was stirred at roomtemperature for 1 hour. The resulting solution was diluted with milliQwater (5 mL) and treated with Amberlite IR-120 (H⁺) to pH 6. The resinwas filtered and washed with milliQ water (15 mL). The filtrate wasconcentrated under reduced pressure to give 23 mg of acid IX (74%) in apale solid yellow form. [α]²⁵ _(D)−121° (c 1.1 in CH₃OH); ¹H-NMR (250MHz, CD₃OD, δ) 8.20 (d, 2H, J 8.8), 7.65 (d, 2H, J 8.8), 5.93 (dd, 1H, J10.0 and 1.8), 5.73 (d, 1H, J 10.0), 4.84 (s, 2H), 4.04 (m, 1H), 3.93(dt, 1H, J 7.9 and 1.8) and 2.07 (m, 2H); ¹³C-NMR (63 MHz, CD₃OD, δ)176.8, 147.6, 147.1, 130.4, 129.3, 128.1, 123.4, 81.1, 73.5, 70.4, 68.2and 40.2; IR (KBr)/cm⁻¹ 3528 and 1596; EM-IQ⁺ (m/z) 310 (MH⁺); HRMScalculated for C₁₄H₁₆NO₇ (MH⁺): 310.0928, 310.0928 found.

EXAMPLE 3

Cyclohexene acids (XIV) were prepared following the second solid phasesynthetic strategy and using carbolactone X, BromoWang polystyrene resinand the corresponding benzyl bromide derivative as starting materials.Thus, treatment of carbolactone I with tetrabutylammonium fluorideprovides alcohol X which, by treatment with sodium hydride andsubsequent reaction with the BromoWang polystyrene resin leads to etherXI. Deprotection of the benzoyl group is carried out by means oftreatment of ether XI with potassium cyanide and gives resin XII incarbolactone XIIa form and methyl ester XIIb form. Treatment of theresin XII with sodium hydride and subsequent reaction with thecorresponding benzyl bromides gives ethers XIII. Finally, the desiredacids XIV are obtained by means of a three-step process consisting firstof breaking the bond with the resin by means of treatment withtrifluoroacetic acid, basic hydrolysis of the lactone and finallytreatment with an ion exchange resin, Amberlite IR-120.

-   -   (a) TBAF, THF, 0° C.; (b) 1. HNa, DMF, 0° C.; 2. BromoWang        resin, Bu₄NI, 15-crown-5, 80° C.; (c) KCN, MeOH, r.t.; (d) 1.        HNa, DMF, 0° C.; 2. R-phenyl-CH₂Br, Bu₄NI, 15-crown-5, 80°        C.; (e) 1. 50% TFA/DCM, 2. LiOH, THF, r.t., 3. Amberlite IR-120        (H⁺).        Preparation of        (1R,3R,4R)-4-benzyloxy-1-hydroxycyclohex-5-ene-1,3-carbolactone        (X).

Tetrabutylammonium fluoride (6.4 mL, 6.36 mmol, 1.0 M commercialsolution in THF) was added to a solution of compound 1 (2.16 g, 5.78mmol) in 80 mL of dry THF under an argon atmosphere and at 0° C. Afterstirring for 30 minutes, it was acidulated with 10% HCl (20 mL) and theorganic phase was extracted with dichloromethane (3×). The pooledorganic phase was dried (Na₂SO₄ anh.), filtered and concentrated underreduced pressure. The obtained residue was purified by means of flashchromatography in silica gel (75% diethyl ether/hexanes) andrecrystallized from hexanes to give 1.38 g of alcohol X in a whitemicrocrystalline form (92%). [α]²⁵ _(D)−103° (c 0.6 in CHCl₃); M.P.104-105° C. (hexanes); ¹H-NMR (250 MHz, CDCl₃, δ) 8.03 (dd, 2H, J 8.5and 1.4), 7.60 (m, 1H), 7.46 (t, 1H, J 7.5), 6.30 (d, 1H, J 9.7), 5.88(ddd, 1H, J 9.7, 3.3 and 1.1), 5.54 (t, 1H, J 3.0), 4.90 (m, 1H), 3.79(broad s, 1H), 2.55 (ddd, 1H J 11.7, 5.2 and 1.7) and 2.49 (d, 1H J11.7); ¹³C-NMR (63 MHz, CDCl₃, δ) 177.1, 165.2, 138.6, 133.7, 129.8,128.9, 128.6, 124.2, 74.3, 73.3, 66.1 and 37.2; IR (KBr)/cm⁻¹ 3478, 1775and 1722; EM-IQ⁺ (m/z) 261 (MH⁺); HRMS calculated for C₁₄H₁₃O₅ (MH⁺):261.0763, 261.0755 found. Analysis calculated for C₁₄H₁₂O₅: C, 64.60; H,4.65. Found: C, 64.60; H, 4.65.

Preparation of Resin XI.

Sodium hydride (264 mg, 6.60 mmol, 60% commercial suspension in mineraloil) was added to a solution of alcohol X (1.43 g, 5.48 mmol) in dry DMF(25 mL) at 0° C. and under an argon atmosphere. After 30 minutes, theresulting suspension was added by means of a cannula to a suspension ofBromoWang polystyrene resin (1.4 g, ˜1.6 mmol/g, ˜2.24 mmol) in dry DMF(16 mL) at 0° C. and under an inert atmosphere. Then 15-crown-5 ether(60 μL, 0.32 mmol) was added and the resulting suspension was gentlystirred at said temperature for 30 minutes and heated at 80° C. for 48hours. The obtained resin was filtered and washed with DMF (3×20 mL),(3:1) DMF/water (3×10 mL), THF (3×10 mL) and dry dichloromethane (2×10mL) to give 1.4 g of resin XI in brown granule form. IR (gel/cm⁻¹) 1794,1718 and 1611; ¹³C-NMR (gel, 63 MHz, CDCL₃, δ) 137.0, 133.6, 129.5,114.6, 73.6, 70.0, 67.5, 40.4 and 34.0.

Preparation of Resin XII.

A solution of potassium cyanide (136 mg, 2.09 mmol) in methanol (2 mL)was added to a solution of resin XI (1.1 g, ˜1.3 mmol) in dry THF (6mL). The resulting solution was bubbled with argon for 2 hours at roomtemperature. The resin was filtered and washed with THF (3×15 mL),methanol (3×15 mL), THF (3×15 mL) and dry dichloromethane (2×10 mL). ˜1g of resin XII was obtained in brown granule form. IR (gel/cm⁻¹) 3392,1792, 1732 and 1604; ¹³C-NMR (gel, 63 MHz, CDCL₃, δ) 129.6, 114.6, 70.2,67.4, 53.4 and 40.4.

The data of some compounds obtained using the processes hereinbeforementioned are indicated below:

(1R,3R,4R)-1-(3′-fluorobenzyloxy)-3,4-dihydroxycyclohex-5-ene-1-carboxylicacid (XIVa).

[α]²⁵ _(D)+11° (c 0.6 in H₂O); ¹⁹F-NMR (282 MHz, D₂O, δ) 111.8 (dt, 1F,J 9.6 and 5.6); ¹H-NMR (250 MHz, D₂O, δ)7.61 (m, 1H), 7.43-7.26 (m, 3H),6.23 (d, 1H, J 10.1), 6.14 (dd, 1H, J 10.1 and 1.8), 4.72 (s, 2H), 4.31(dt, 1H, J 8.2 and 1.7), 4.08 (ddd, 1H, J 12.2, 3.7 and 8.2), 2.44 (ddd,1H, J 13.6, 3.0 and 1.1) and 2.19 (dd, 1H, J 13.6 and 12.2); ¹³C-NMR (63MHz, D₂O, δ) 175.2, 162.9 (J 242), 140.6 (J 7), 134.6, 130.6 (J 8),127.2, 124.5 (J 3), 115.4 (J 21), 115.1 (J 21), 80.0, 72.7, 69.6, 67.1and 38.2; IR (KBr)/cm⁻¹ 3400, 1716 and 1592; EM-IQ⁺ (m/z) 265 (MH⁺-H₂O);HRMS calculated for C₁₄H₁₄O₄F (MH⁺): 265.0876, 265.0870 found.

(1R,3R,4R)-3,4-dihydroxy-1-(4′-cyano)benzyloxycyclohex-5-ene-1-carboxylicacid (XIVb).

M.P. 81-82° C.; [α]²⁵ _(D)+18° (c 0.6 in CH₃OH); ¹H-NMR (250 MHz, CD₃OD,δ) 7.66 (d, 2H, J 8.3), 7.55 (d, 2H, J 8.3), 6.01 (d, 1H, J 10.1), 5.87(dd, 1H, J 10.1 and 2.0), 4.64 (d, 1H, J 12.3), 4.56 (d, 1H, J 12.3),3.98 (dt, 1H, J 7.8, 2.0 and 1.8), 3.86 (ddd, 1H, J 7.8, 11.5 and 3.6),2.26 (ddd, 1H, J 13.3, 2.2 and 3.6) and 1.98 (dd, 1H, J 11.5 and 13.3);¹³C-NMR (63 MHz, CD₃OD, δ) 177.2, 146.4, 135.7, 133.1, 129.3, 128.3,119.8, 111.9, 80.8, 74.1, 70.9, 67.1 and 39.7; IR (KBr)/cm⁻¹ 3410, 2232and 1716; EM-IQ⁺ (m/z) 272 (MH⁺-H₂O); HRMS calculated for C₁₅H₁₄NO₄(MH⁺): 272.0923, 272.0930 found.

(1R,3R,4R)-1-(4′-fluorobenzyloxy)-3,4-dihydroxycyclohex-5-ene-1-carboxylicacid (XIVc).

[α]²⁵ _(D)+9° (c 1.0 in H₂O); ¹⁹F-NMR (282 MHz, D₂O, δ) 112.6 (tt, 1F, J9.1 and 5.2); ¹H-NMR (250 MHz, D₂O, δ) 7.32 (dd, 2H, J 7.7 and 5.7),7.04 (t, 2H, J 8.7), 5.91 (m, 2H), 4.40 (s, 2H), 4.03 (dd, 1H, J 8.2 and1.2), 3.78 (ddd, 1H, J 11.6, 8.2 and 3.45), 2.15 (dd, 1H, J 13.6 and3.45) and 1.90 (t, 1H, J 13.6 and 11.6); ¹³C-NMR (63 MHz, D₂O, δ) 177.8,162.7 (J 242), 134.8, 133.8, 131.1 (J 8), 127.0, 115.6 (J 21), 80.3,72.7, 69.6, 67.2 and 38.0; IR (KBr)/cm⁻¹ 3420, 1716 and 1605; EM-IQ⁺(m/z) 265 (MH⁺-H₂O); HRMS calculated for C₁₄H₁₄O₄F (MH⁺): 265.0876,263.0880 found.

1. Compounds of formula (1), comprising:

R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ can be a hydrogen atom, an acyloxy,alkyloxy, aryloxy, alkylthio, alkylamine, alkylnitro, alkylazido,alkylphosphate, alkylcarboxy, arylthio or alkyl group with C1-C10 chainsor a benzyloxy group in which the aromatic ring can be substituted byone or several identical or different radicals, chosen from halogen,polyhalogenated alkyl, nitro, azido, amino, phosphate, carboxy, cyano,amide, thiol, thioester, guanidinium, thioether, alcohol, alcoxy oralkyl group with C1-C10 chains. The radicals can be linear or branchedalkyl with 1-10 carbon atoms, alkenyl with 2 to 10 carbon atoms, alkynylwith 3 to 10 carbon atoms, cycloalkyl with 3 to 6 carbon atoms,cycloalkenyl with 4 to 6 carbon atoms, or bicycloalkyl with 7 to 10carbon atoms; these radicals possibly being substituted by one orseveral identical or different substituents chosen from halogen atomsand hydroxy, amino, thiol, azido, nitro, phosphate and alkoxy radicalscontaining 1 to 4 carbon atoms, piperidinyl, morpholinyl, indole, furan,piperazinyl-1 (possibly substituted at −4 by an alkyl radical with 1 to4 carbon atoms or by a phenylalkyl radical, the alkyl part of whichcontains 1 to 4 carbon atoms), cycloalkyl with 3 to 6 carbon atoms,cycloalkenyl with 4 to 6 carbon atoms, phenyl, cyano, nitro, carboxy,alkoxycarbonyl, halogen, amino or amide, the alkyl part of whichcontains 1 to 4 carbon atoms, or a phenyl radical, possibly substitutedby one or several identical or different radicals, chosen from the alkylradicals with 1 to 4 carbon atoms, halogenated or not, or alkoxyradicals with 1 to 4 carbon atoms, or halogen, nitro, azido, phosphate,amino, cyano, amide, thiol, thioester, guanidinium, thioether or alcoholgroups, a saturated or unsaturated nitrogenous heterocyclic radicalcontaining 1 to 4 carbon atoms, a saturated or unsaturated nitrogenousheterocyclic radical containing 5 or 6 members, possibly substituted byone or several alkyl radicals with 1 to 4 carbon atoms, understandingthat the cycloalkyl, cycloalkenyl or bicycloalkyl radicals can possiblybe substituted by one or several alky radicals containing 1 to 4 carbonatoms.
 2. A process (process 1) for preparing the compounds of formula(1), characterized, as the most important synthetic transformations, bythe following steps:

a) alkylation of the alkoxide carrying the cyclohexane ring of generalformula (2),

with electrophilic resins of general formula (3),

obtaining compounds of general formula (4) as a reaction product,wherein the R¹, R², R⁵, R⁶, R⁸, R¹⁰ and R¹¹ groups have the structuralcharacteristics indicated in claim 1, and the X group can be a halogen,a sulfonate group, any other leaving group or a carbonyl group:

b) alkylation of the compounds of general formula (4) from the previousstep a) in an inert solvent so as to obtain ethers of general formula(5),

wherein the R¹, R², R⁵, R⁶, R¹⁰ and R¹¹ groups have the characteristicsdescribed hereinbefore, and the R⁷group has the structuralcharacteristics indicated in claim 1; c) resin cleaving reactionfollowed by lactone hydrolysis, obtaining products of general formula(6),

wherein the R¹, R², R⁵, R⁶ and R⁷ groups have the meaning hereinbeforegiven; d) subsequent modifications of functional groups such asoxidations, reductions, esterifications, alkylations, isomerizations,etc., to give the compounds of general formula (1)

wherein the R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ groups have the meaningpreviously given in claim
 1. 3. A process (process II) for preparing thecompounds of formula (1), characterized, as the most important synthetictransformations, by the following steps:

a) alkylation of the alkoxide carrying the cyclohexane ring of generalformula (7),

with electrophilic resins of general formula (3), previously describedin claim 2, obtaining compounds of general formula (8) as a reactionproduct, wherein the R¹, R², R⁵, R⁶, R¹⁰, R¹¹ and R¹² groups have thestructural characteristics indicated in claim 1, and the X group has thecharacteristics indicated in claim 2:

b) alkylation of the compounds of general formula (8) from the previousstep a) in an inert solvent so as to obtain ethers of general formula(9),

wherein the R¹, R², R⁵, R⁶, R¹⁰ and R¹¹ groups have the characteristicsdescribed hereinbefore, and the R³group has the structuralcharacteristics indicated in claim 1; c) resin cleaving reactionfollowed by lactone hydrolysis, obtaining products of general formula(10),

wherein the R¹, R², R³, R⁵ and R⁶ groups have the meaning hereinbeforegiven; d) subsequent modifications of functional groups, such asoxidations, reductions, esterifications, alkylations, isomerizations,etc., to give the compounds of general formula (1)

wherein the R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ groups have the meaningpreviously given in claim
 1. 4. A process (process III) for preparingthe compounds of formula (1), characterized, as the most importanttransformations, by the following steps:

a) alkylation of the alkoxide carrying the cyclohexene ring of generalformula (7), with electrophiles of general formula (11),

obtaining compounds of general formula (12) as a reaction product,wherein the R¹, R², R⁵, R⁶, R¹², R¹³, R¹⁴ and R¹⁵ groups have thestructural characteristics indicated in claim 1, and the X group can bea halogen, a sulfonate group, any other leaving group or a carbonylgroup:

b) lactone hydrolysis reaction, obtaining products of general formula(13)

wherein the R¹, R², R³, R⁵, R⁶, R ³, R¹⁴ and R¹⁵ groups have the meaninghereinbefore given; c) subsequent modifications of functional groups,such as oxidations, reductions, esterifications, alkylations,isomerizations, etc., to give the compounds of general formula (1)

wherein the R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ groups have the meaningpreviously given in claim
 1. 5. A process (process IV) for preparing thecompounds of formula (1), characterized, as the most importanttransformations, by the following steps:

a) alkylation of the alkoxide carrying the cyclohexene ring of generalformula (2) with electrophiles of general formula (11), respectively,obtaining compounds of general formula (14) as a reaction product,wherein the R¹, R², R⁵, R⁶, R⁸, R¹³, R¹⁴ and R¹⁵ groups have thestructural characteristics indicated in claim 1,

b) lactone hydrolysis re action, obtaining product s of general formula(15)

wherein the R¹, R², R⁵, R⁶, R⁷, R¹³, R¹⁴ and R¹⁵ groups have the meaninghereinbefore given; c) subsequent modifications of functional groups,such as oxidations, reductions, esterifications, alkylations,isomerizations, etc., to give the compounds of general formula (1)

wherein the R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ groups have the meaningpreviously given in claim
 1. 6. A pharmaceutical compositioncharacterized in that it contains a compound of claim 1 as an activeingredient in a mixture with the suitable vehicle or carrier.
 7. Use ofthe compounds of the formula of claim 1 in the production of anantitumor pharmaceutical composition.
 8. Use of the compounds of theforumla of claim 1 in the production of an antifungal pharmaceuticalcomposition.
 9. Use of the compounds of the formula of claim 1 in theproduction of an antimicrobial pharmaceutical composition.
 10. Use ofthe compounds of the formula of claim 1 in the production of anantiviral pharmaceutical composition.
 11. Use of the compounds of theformula of claim 1 in the production of an immunosuppressantpharmaceutical composition.
 12. Use of the compounds of the formula ofclaim 1 in the production of an herbicidal composition.